Penicilliumdigitatum is the most destructive postharvest pathogen of citrus fruits, causing fruit decay and economic loss. Additionally, control of the disease is further complicated by the emergence of drug-resistant strains due to the extensive use of triazole antifungal drugs. In this work, an orthologus gene encoding a putative sterol regulatory element-binding protein (SREBP) was identified in the genome of P. digitatum and named sreA. The putative SreA protein contains a conserved domain of unknown function (DUF2014) at its carboxyl terminus and a helix-loop-helix (HLH) leucine zipper DNA binding domain at its amino terminus, domains that are functionally associated with SREBP transcription factors. The deletion of sreA (ΔsreA) in a prochloraz-resistant strain (PdHS-F6) by Agrobacteriumtumefaciens-mediated transformation led to increased susceptibility to prochloraz and a significantly lower EC50 value compared with the HS-F6 wild-type or complementation strain (COsreA). A virulence assay showed that the ΔsreA strain was defective in virulence towards citrus fruits, while the complementation of sreA could restore the virulence to a large extent. Further analysis by quantitative real-time PCR demonstrated that prochloraz-induced expression of cyp51A and cyp51B in PdHS-F6 was completely abolished in the ΔsreA strain. These results demonstrate that sreA is a critical transcription factor gene required for prochloraz resistance and full virulence in P. digitatum and is involved in the regulation of cyp51 expression. P. digitatum is the most destructive disease of citrus fruit, responsible for up to 90% of total crop losses during postharvest packing, storage, transportation, and marketing carbamoyl}; PRC) for the different strains were measured according to [6 spores ml-1) of the HS-F6 wild-type and mutant strains was coated onto a PDA plate respectively and cultivated at 25°C for 24 h. Mycelial plugs (approximately 0.8 mm diameter) were then obtained from the plate using a punch and placed on the center of PDA plates containing different concentrations of prochloraz. After incubation at 25°C for 6 days, the diameters of the colonies were measured. Three replicates were used for each experiment. The average of the colony diameters in each independent test was used for EC50 calculation by software SPSS 10.0.ECrding to  with modCitrus sinensis) were purchased from a fruit market in Hongshan district, Wuhan. The fruits were washed with distilled water and dried at room temperature before inoculation. Virulence assays for the HS-F6 and mutant strains were performed directly on citrus fruits. Firstly, a 2-mm deep hole was made on the pericarp using a 1-ml pipett tip. Then 3 μl of a conidial suspension (106 spores ml-1) of the HS-F6 wild-type or mutants was injected into the hole. After incubation at 25°C for three days, the diameters of the disease spots formed were measured and compared.Mature citrus fruits  of P. digitatum HS-F6 and ΔsreA strains was cultured in PDB medium at 25°C for 72 h. In the prochloraz-treatment experiment, 7 μg/ml prochloraz (about the concentration of EC50) was added to the PDB medium with shaking for an extra 6 h after cultured at 25°C for 48 h. The mycelia were then filtered and washed several times using double distilled water. Total RNA was extracted using RNAiso Plus  according to the manufacturer’s protocol. All RNA samples were treated with DNase I . First-strand cDNA was prepared using All-in-one First strand cDNA Synthesis Kit  following the manufacturer’s protocol. qRT-PCR was performed using a BIO-RAD CFX96 q-PCR system with SYBR Green I fluorescent dye detection. The mRNA abundance was normalized using the housekeeping gene β-actin, and the relative expression levels were calculated using the 2-ΔΔCt method [cyp51A/B/C and β-actin genes in qRT-PCR are listed in Table 1.qRT-PCR was used to analyze t method . The priP<0.05 was considered significant.All results presented with statistical significance were analyzed with an unpaired two-tailed Student’s t test and two-way ANOVA. sreA ORF was amplified from PdHS-F6 genomic DNA using primers sreA-F and sreA-R (Table 1), sequenced, and then analyzed by NCBI BLAST. The sreA ORF is predicted to encode a protein of 1040 amino acids that is a putative HLH transcription factor and an ortholog of A. fumigatus transcription factor SrbA. The nucleotide homology between sreA and srbA is 63%; protein homology is 61% . The nucleotide homology between sreA and P. chrysogenum Pc20g05880 is 89%.A 3120-bp DNA fragment of Two highly conserved domains in SreA were identified. A basic helix-loop-helix-leucine zipper (bHLH-zip) domain located in the N-terminus of the protein contains a unique tyrosine residue (at site 177) that distinguishes SREBPs from other bHLH transcriptional factors ; a domaiP. digitatum, sreA disruption strains (ΔsreA) of HS-F6 (PdHS-F6m) were constructed . Twenty putative mutants were selected on PDA medium supplemented with 50 μg/ml hygromycin B and screened by PCR using primers sreA-1 and sreA-4 (Table 1). The 3.0-kb fragment of sreA in HS-F6 was replaced by a 4.0-kb fragment (the sreA gene disrupted with the hph gene) in the recombinant strains (ΔsreA) . Southern blot analysis further confirmed that a single copy was integrated. Only one strain was chosen and used for further study .To analyze the function of SreA in sreA) were obtained using G418 as selective marker. The insertion of sreA in the COsreA strains was confirmed by PCR using primers sreA-1 and sreA-4. The occurrence of a 3.0-kb band (sreA) and a 4.0-kb band (sreA disrupted with the hph) indicated the successful integration of the sreA gene into the genome of the ΔsreA strains . Southern blotting confirmed that the ΔsreA and COsreA strains were successfully constructed and that a single copy of sreA was integrated into the genome of the ΔsreA strain to generate the COsreA strain .Twenty complementation strains  were smaller than those of HS-F6. After being cultured on PDA plates supplemented with different concentration of prochloraz  for 6 days, the diameters of the colonies were measured , and the EC50 value was calculated. The average EC50 value of prochloraz for the ΔsreA strain was 3.2 mg/l, which was less than half of the value of HS-F6 . However, complementation of sreA (PdHS-F6c) restored the EC50 value of the ΔsreA strain . These results demonstrated that the deletion of sreA renders P. digitatum more susceptible to triazole drug prochloraz, suggesting that sreA plays an important role in the resistance of P. digitatum to triazole drugs.As stated above, PdHS-F6 is highly-resistant to triazole drug prochloraz and has an ECsreA plays a role in the virulence of P. digitatum toward citrus fruits, a virulence assay was performed directly on these fruits. The assay results demonstrated that the symptoms in the fruits incubated with conidial suspension of the ΔsreA strains developed more slowly than in the fruits incubated with the wild-type conidial suspension. The mean diameter of the macerated lesions of the fruits incubated with the ΔsreA conidial suspension was approximately 1.90 cm at 3 days post inoculation, whereas the mean macerated lesion diameter of the fruits incubated with the HS-F6 conidial suspension was about 3.4 cm . The virulence assay results revealed that the deletion of sreA rendered the ΔsreA strain less virulent compared with the wild-type HS-F6. A further experiment was performed to confirm this result. The virulence assay demonstrated that average diameter of the macerated lesions induced by COsreA was comparable to that of wild type HS-F6 (3.2 cm) . These results indicated that SreA is required for full virulence in P. digitatum.To determine whether erg11A gene in A. fumigatus [P. digitatum. To test this hypothesis, the mRNA abundance of the cyp51 genes in PdHS-F6 wild-type and the ΔsreA strain was analyzed using qRT-PCR . The results showed that transcriptional abundance of the three cyp51 genes were all decreased in the ΔsreA strain, especially with regard to cyp51A. The normalized expression values of cyp51A, cyp51B, and cyp51C in the ΔsreA strain were 0.02, 0.29 and 0.47, respectively.Because SrbA regulates the expression of umigatus , we hyposreA in the transcriptional response of P. digitatum to prochloraz, a prochloraz induction experiment was performed with the ΔsreA and HS-F6 strains. Total RNA was isolated from the PdHS-F6 and ΔsreA strains with or without prochloraz treatment and used for qRT-PCR. In the case of wild-type HS-F6, the expression levels of the cyp51 genes were all increased after 6 h of prochloraz treatment ; the normalized expression values in the cyp51A, cyp51B and cyp51C expression levels after prochloraz treatment were 5.3, 1.8, and 2.2, respectively. However, the induction of cyp51A and cyp51B by prochloraz was abolished in the ΔsreA strain. As shown in Fig. 6B, the normalized expression values for cyp51A and cyp51B mRNA abundances were 0.56 and 0.65, respectively. Notably, the increasing fold-change in cyp51C mRNA abundance after prochloraz treatment was higher in the ΔsreA strain than in the wild-type strain HS-F6 , which might be due to regulation by other transcription factors that maintained cyp51C transcript level in response to prochloraz.To investigate the role of S. cerevisiae and C. albicans, was the first documented transcription factor highly correlated with fungal drug resistance [CaUpc2 rendered C. albicans increased susceptibility to a range of common antifungal drugs used in clinical therapy, and this increased susceptibility is observed for drugs targeting the ergosterol biosynthesis pathway [C. albicans erg genes involved in ergosterol biosynthesis, including erg7, erg11 and erg25 [erg11 overexpression in the Upc2 gain-of-function mutants led to strain resistance to azole fungicides [S. pombe as well as a number of fungal pathogens particularly grown under hypoxia conditions [A. fumigatus. It is notable that SrbA plays a critical role for triazole drug interactions in A. fumigates, which may have clinical importance [S. cerevisiae and C. albicans, sterol synthesis is regulated by Upc2 instead [A number of transcription factors associated with sterol metabolism and drug resistance have been well characterized in different clinical fungi. Upc2, a typical zinc cluster protein identified in sistance –37. Dele pathway . A prevind erg25 . The ergngicides . SREBPs,nditions , 40. Srenditions –42. SrbAportance –24. In m instead . Even thP. digitatum as an ortholog of SrbA that is associated with triazole susceptibility, full virulence, and regulation of cyp51 genes [P. digitatum may play a role similar to SrbA. The sreA deletion strains of P. digitatum (ΔsreA) were generated using A. tumefaciens-mediated genetic transformation based on the prochloraz-resistant P. digitatum strain PdHS-F6. Prochloraz is a triazole fungicide that is widely used in Europe, Australia, Asia and South America within gardening and agriculture [P. digitatum were isolated from citrus fruits collected in Hubei Province and 25 isolates were identified to be prochloraz-resistant, with a proportion as high as 32% [In the present study, we characterized an SREBP transcriptional factor, SreA, in 51 genes . Our resiculture . Howeverh as 32% . For redP. digitatum demonstrated that the deletion of sreA increases susceptibility of PdHS-F6 to prochloraz. As shown in Fig. 3, the prochloraz EC50 value of the ΔsreA strain was significantly lower than HS-F6. The growth assay on PDA plates revealed that the ΔsreA strain grew as well as PdHS-F6 without prochloraz. However, the growth rate of ΔsreA strain on PDA plates supplemented with prochloraz  was much slower than that of the HS-F6 , revealing that drug response was altered in the ΔsreA strain. CYP51 proteins are the targets of triazole fungicides, and the susceptibility of fungi to triazole drugs is closely related with the mutations in or the expression level of cyp51 genes [cyp51 gene expression, the transcriptional abundance of cyp51 genes in HS-F6 and ΔsreA strain were analyzed by qRT-PCR. The results revealed that the expression level of the three cyp51 genes was decreased in the ΔsreA strain, particularly cyp51A, which almost could not be detected . Our study thus demonstrated that SreA is an important regulator of cyp51 genes: the deletion of sreA reduced the expression levels of cyp51 genes, which are the targets of trizole fungicide prochloraz, rendering the strain more susceptible to the drug. However, SreA is not the only transcription factor regulating the expression of cyp51 genes. In the ΔsreA strain, other regulators may control the expression of cyp51 genes in response to the drug and to some extent may compensate the loss of sreA. In support of this notion, the EC50 value of the ΔsreA strain was not as low as the prochloraz-susceptible strain, and the ΔsreA strain could still grow on PDA plates supplemented with prochloraz at a relatively high concentration; nevertheless, the growth rate was much slower than that of HS-F6.Our results in 51 genes . To confsreA is associated with pathogenicity of P. digitatum, virulence assays were performed directly on citrus fruits using the HS-F6 wild-type and mutant strains. The results demonstrated that the ΔsreA strain was less virulent than the wild type. The symptoms in fruits incubated with a ΔsreA conidial suspension developed more slowly and the mean diameter of the macerated lesions was almost half of those produced by the HS-F6 wild-type strain . As expected, the COsreA strain displayed symptoms similar to the wild type, indicating that the complementation of sreA restored the virulence of ΔsreA strain. Therefore, we can conclude that SreA is required for full virulence in P. digitatum.To determine whether erg25 transcription is induced in response to itraconazole treatment in C. albicans [cyp51 genes in fungi has been regarded as an important fungal self-protective mechanism towards a range of fungicides [cyp51 mRNA expression in PdHS-F6 and ΔsreA strains before and after prochloraz treatments. In the HS-F6 wild-type strain, the expression level of cyp51 genes were all increased after prochloraz induction for 6 h . However, the increase in cyp51 expression levels, except cyp51C, was abolished in the ΔsreA strain. The further increased mRNA abundance of cyp51C in response to prochloraz-treatment in the ΔsreA strain might be attributed to the regulating by other transcription factors in response to the drug. However, the relatively decreased expression level of cyp51C in the ΔsreA strain demonstrated that sreA could regulate the expression of cyp51C to some extent. These results indicated that SreA is an important regulator of cyp51 genes. The deletion of sreA renders P. digitatum unable to cope with prochloraz properly, making it more susceptible to the antifungal drug.Fungi have evolved sophisticated mechanisms to cope with environmental stresses, including antifungal drugs, and respond to triazole drugs by altering the expression levels of effector genes. DMI-resistant isolates were initially thought to exhibit fitness penalties that would preclude DMI resistance and becoming wide-spread in nature, as reported in the studies of several important fungal pathogens of different crops . In the albicans . Besidesngicides . In thiscyp51 genes or the mutation of their regulatory genes [C. albicans, tumorigenesis in hosts, and for resistance of the malarial parasite Plasmodium vivax [cyp51 from P. digitatum and Ustilago maydis [P. digitatum [Ustilago maydis [in vivo [Thus, as a regulator of cyp51, SreA, with an essential function in P. digitatum fungicide resistance, represents a promising research direction to uncover a new fungus-specific antifungal drug targets.With the widespread and constant use of triazloe antifungal drugs, resistance has been selected in many fungal species. Most of this resistance is due to the mutation and overexpression of target ry genes , 18–19. ry genes . The docum vivax –51. Thero maydis –55. The igitatum . The scr[in vivo . Thus, a


It has recently been shown that the wear of ultra-high-molecular-weight polyethylene in hip and knee prostheses leads to the generation of nanometre-sized particles, in addition to micron-sized particles. The biological activity of nanometre-sized ultra-high-molecular-weight polyethylene wear particles has not, however, previously been studied due to difficulties in generating sufficient volumes of nanometre-sized ultra-high-molecular-weight polyethylene wear particles suitable for cell culture studies. In this study, wear simulation methods were investigated to generate a large volume of endotoxin-free clinically relevant nanometre-sized ultra-high-molecular-weight polyethylene wear particles. Both single-station and six-station multidirectional pin-on-plate wear simulators were used to generate ultra-high-molecular-weight polyethylene wear particles under sterile and non-sterile conditions. Microbial contamination and endotoxin levels in the lubricants were determined. The results indicated that microbial contamination was absent and endotoxin levels were low and within acceptable limits for the pharmaceutical industry, when a six-station pin-on-plate wear simulator was used to generate ultra-high-molecular-weight polyethylene wear particles in a non-sterile environment. Different pore-sized polycarbonate filters were investigated to isolate nanometre-sized ultra-high-molecular-weight polyethylene wear particles from the wear test lubricants. The use of the filter sequence of 10, 1, 0.1, 0.1 and 0.015 µm pore sizes allowed successful isolation of ultra-high-molecular-weight polyethylene wear particles with a size range of < 100 nm, which was suitable for cell culture studies. Even though the nanometre-sized UHMWPE wear particles occupy a relatively small percentage of the total wear volume, they are produced in large numbers. Due to their small size, numerous nanometre-sized UHMWPE wear particles may be disseminated throughout the body and UHMWPE wear particles have previously been found in the lymph nodes, spleen and liver.1214 Therefore, it is essential to determine the relative contribution of nanometre-sized UHMWPE to the inflammatory process in osteolysis of THRs. Currently, however, there are no reports on the biological response to nanometre-sized UHMWPE wear particles, due to the difficulty in obtaining sufficiently large volumes of nanometre-sized UHMWPE wear particles for in vitro studies.Ultra-high-molecular-weight polyethylene (UHMWPE) wear particles have been shown to play a major role in late aseptic loosening of conventional total hip replacements (THRs).15 It is therefore critical that any contaminating endotoxin is removed from wear particles prior to cell culture studies.Endotoxin is a major constituent of gram-negative bacterial cell walls, which easily adheres to polyethylene particles. Endotoxin stimulates macrophages to produce nitric oxide (NO), cytokines, such as tumour necrosis factor-α (TNF-α), and interleukins (ILs), such as IL-6.16 Endotoxin is highly heat stable, and standard autoclaving at 121 °C is not able to destroy endotoxin. It has been reported that endotoxin can be destroyed by heating to 250 °C for more than 30 min or 180 °C for more than 3 h; however, polyethylene particles that are a type of polymer material cannot withstand these conditions.17Endotoxin contamination is not only prevalent in the environment such as air, tap water and an individual’s skin, but also exists in laboratory environments, such as chemical reagents, buffers, raw materials, glassware and equipment.19 Although these studies provided efficient methods for removing endotoxin from UHMWPE wear particles, loss of particles after the treatment could not be avoided.Several studies have investigated different methods of removing endotoxin from UHMWPE wear particles using either chemical washing or ultracentrifugation methods.20 Therefore, generating UHMWPE wear particles in a sterile environment, which would remove any potential source of endotoxin, provided a promising way to generate endotoxin-free particles while also avoiding loss of particles. The aims of this study were to investigate different pin-on-plate simulation methods for generation of a large volume of endotoxin-free clinically relevant nanometre-sized UHMWPE wear particles suitable for cell culture studies.In our previous studies, it has been shown that it is possible to generate endotoxin-free, clinically relevant UHMWPE wear particles in a lubricant of RPMI 1640 supplemented with 25% (v/v) foetal calf serum using a single-station pin-on-plate simple configuration wear simulator under aseptic conditions.GUR 1020 UHMWPE pins  were sterilised using gamma irradiation in a vacuum (2.5–4 mrad). High-carbon  cobalt–chromium alloy (CoCr) plates  had a surface roughness (Ra) of 0.07–0.08 µm.20 RPMI 1640 medium  was used as a lubricant for the wear test. Metal components (stainless steel) of the wear rig and CoCr plates were sterilised by dry heat at 190 °C for 4 h, and the polymer parts (polypropylene) were sterilised by autoclaving at 121 °C for 20 min at 103 kPa pressure prior to the test. The test was run at 1 Hz, with a stroke length of 28 mm and a rotation of ±30°. A load of 160 N was applied to produce a nominal contact pressure of 3.2 MPa. The wear tests were run for a total of 14 weeks. The sterility of the RPMI 1640 medium lubricant was evaluated daily by microbiological testing. Post-test lubricants containing UHMWPE wear particles were collected and stored at −20 °C for isolation of particles.The multidirectional single-station pin-on-plate wear simulator was assembled using aseptic technique and run within a class II safety cabinet . Three filter sequences were investigated, including (A) 10, 1, 0.1 and 0.015 µm filters; (B) 10, 1, 0.1, 0.05 and 0.015 µm filters and (C) 10, 1, 0.1, 0.1 and 0.015 µm filters. Prior to filtration, the wear test lubricants were subjected to a minimum of 40 min of sonication in an ultrasonic water bath (Grant Instruments (Cambridge) Ltd, Royston, Herts, UK) to limit agglomeration of wear particles. After filtration, the final 0.015 µm filters were dried under an infrared heat lamp for 4 h in a moisture-controlled room. The filters were then weighed a minimum of five times on a Mettler Toledo AT 21 digital microbalance  with a measuring accuracy of ±5 µg. The mass of the nanoscale particles was calculated.A small section of the 0.015 µm filter was cut and mounted onto an aluminium stub and sputter coated with platinum/palladium  to a thickness of 3.0 nm, which eliminated electron charging of the particles. The particles on the 0.015 µm filters were characterised using high-resolution field emission gun-scanning electron microscopy  images.Particles were analysed manually using Image-Pro Plus software , and the length of the particles and their area were measured. A minimum of 100 particles were analysed for each sample using a range of magnifications from 10,000× to 200,000×. For samples with a large number of nanometre-sized particles, more than 500 particles were measured for each image. The data were exported to an Excel spreadsheet and grouped into different size ranges according to the length of the particles. The area of the image was recorded (A), and the number of particles in each size range was recorded (N). P represented the sum of the areas of the particles in each size range. Values of the number of particles per area (N/A) and the average area of particles (P/N) for each size range within each field of view were calculated. The percentage number of particles and percentage area distribution of particles in each size range were generated from these data for illustrative purposes.Energy-dispersive X-ray analysis  was used to determine the elemental composition of the particles of all morphologies on the 0.015 µm  filters. EDX allowed identification of contaminants; however, it is not a method that allows positive identification of UHMWPE, since the UHMWPE particles contain the elements carbon, hydrogen and oxygen, which are also present in the polycarbonate filter. Therefore, particles, which showed main element peaks including carbon and oxygen on the EDX trace, were considered to be UHMWPE.Microbiological tests were performed by plating lubricant samples on heated blood agar (HBA), nutrient agar (NA) and Sabouraud dextrose agar (SAB). The HBA and NA plates were incubated at 37 °C, and the SAB plates were incubated at 30 °C for 24 h. A volume of 1 mL of lubricant sample was transferred into 10 mL of nutrient broth and incubated with shaking at 37 °C for a minimum of 2 weeks. All microbiological culture media were supplied by the Media Lab, Institute of Molecular and Cell Biology, University of Leeds, Leeds, UK.The pyrochrome Limulus amebocyte lysate (LAL) kinetic-QCL endotoxin assay  was used to determine the presence of endotoxin in the wear test lubricants and fractionated nanometre-sized particle stocks. The assay was performed according to the manufacturers’ instructions.The lubricants obtained from the first 3 weeks of wear testing were filtered through a filter sequence A , and particles were collected on the 0.015 µm filter. The morphologies of particles isolated from filter sequence A are shown in For particles isolated from filter sequence A, although 90.19% of the particles were less than 100 nm in size, particles with a size range of greater than 100 nm accounted for a high percentage area of particles 22.07%; . For parThe lubricants from six-station wear simulation were filtered through the filter sequence C . The morphologies of particles obtained on the final 0.015 µm filters are shown in 21All of the wear tests performed in the single-station and six-station pin-on-plate simulators were free from microbiological contamination. The six-station lubricants and isolated nanometre-sized particle stocks showed very low endotoxin levels, which were below the accepted value of less than 5 endotoxin units (EU) according to the specification of endotoxin tolerance limit for non-pyrogenic products for the pharmaceutical industry.The main peaks for the polycarbonate filter were carbon and oxygen. Trace elements of platinum and silicon were also present at very low levels, as shown in −9. Particles that are less than 100 nm in size have been defined as being in the nanometre-sized range in previous studies.36 Therefore, the aim of this study was to generate only nanometre-sized UHMWPE wear particles, that is, those smaller than 100 nm, in isolation.The prefix ‘nano’ is used in science to mean 1 × 1022 while other factors, such as molecular weight, composition, surface texture and shape, have also been shown to stimulate different cellular responses.24 Previous studies26 have used commercially available UHMWPE particles, which were convenient and cost-effective; however, the particles were almost spherical in shape and had quite different morphologies from particles isolated from retrieved tissues of patients with failed total hip prostheses, which were more varied in shape and had irregular surface textures. Therefore, it is important to generate particles by wear simulation to ensure that they are representative of clinically generated particles.The size and volume concentration of particles have been shown to be the most important determinants in cell responses to UHMWPE particles,6 which indicated that the nanometre-sized UHMWPE wear particles generated in this study were clinically relevant particles. The use of the single-station wear simulators solved two of the problems in generating clinically relevant sterile particles; however, a mass of only 47 µg of nanometre-sized particles, which was equivalent to 0.0183% (w/w) of the total wear loss, was obtained after 8 weeks of wear testing, indicating that it was not practical to generate a large volume of UHMWPE wear particles using the single-station wear simulator.Generating large volumes, sterile, clinically relevant particles were three key problems in this study. The single-station pin-on-plate simulator was used initially to generate clinically relevant nanometre-sized UHMWPE wear particles under aseptic conditions. Microbiological tests showed that none of the wear tests experienced bacterial contamination. In order to obtain particles with a size of less than 100 nm, three different filtration sequences were investigated. Filter sequence C, which comprised 10, 1, 0.1, 0.1 and 0.015 µm filters, generated the lowest percentage area of particles greater than 100 nm (0.048%) and produced particles only within the nanometre-sized range (<100 nm). This filter sequence represented the optimised size distribution for isolating nanometre-sized UHMWPE wear debris. Furthermore, the agglomerated granular morphologies of the nanometre-sized UHMWPE particles isolated were extremely similar to those from retrieved tissues,Therefore, the feasibility of using a six-station multidirectional pin-on-plate wear simulator was investigated, which was expected to produce six times the amount of wear compared to the single-station wear simulator. As the six-station wear simulator could not be housed in a sterile environment, it was a challenge to generate endotoxin-free wear particles in a non-sterile environment. However, as a result of careful set-up and operation and the omission of serum from the RPMI 1640 medium lubricant, there was no microbiological contamination of the lubricants, and the isolated nanometre-sized UHMWPE wear particles showed acceptable endotoxin levels, which indicated that it was feasible to generate endotoxin-free wear particles in a non-sterile environment.4 reported, for the first time, on the isolation of nanometre-sized UHMWPE particles down to 10 nm from the lubricants retrieved from hip and knee simulators. Richards et al.5 identified nanometre-sized UHMWPE particles from tissue samples taken from failed Charnley THRs. The smallest particle identified in vivo was 30 nm. Recently, Lapcikova et al.6 isolated nanometre-sized UHMWPE wear particles with a size of less than 50 nm from periprosthetic (granuloma) tissues obtained during revision of total joint replacements. In this study, nanometre-sized wear particles with a size range below 10 nm were identified in the lubricants from six-station pin-on-plate wear simulation  of the total wear loss), which indicated that the use of the six-station multidirectional pin-on-plate wear simulator achieved the aim of obtaining a large volume of sterile clinically relevant UHMWPE wear particles, which would be suitable for cell culture studies to investigate the biological response to nanometre-sized UHMWPE wear particles.A multidirectional pin-on-plate six-station wear simulator provided an efficient way to generate a large volume of endotoxin-free, clinically relevant nanometre-sized UHMWPE wear particles in vitro in order to investigate the contribution of nanoscale wear debris to osteolysis inflammation and adverse biological responses.


Here we report that a Group 4 catalase, catalase-phenol oxidase (named as AcCATPO), was identified, purified and characterized from leaves of Amaranthus cruentus, a betalain plant. The purified enzyme appeared to be a homotrimeric protein composed of subunits of about 58 kDa, and demonstrated not only the catalase activity toward H2O2, but also the monophenolase activity toward L-tyrosine and diphenolase activity toward L-DOPA. Its catalase and phenol oxidase activities were inhibited by common classic catalase and tyrosinase inhibitors, respectively. All its peptide fragments identified by nano-LC-MS/MS were targeted to catalases, and matched with a cDNA-encoded polypeptide which contains both classic catalase and phenol oxidase active sites. These sites were also present in catalases of non-betalain plants analyzed. AcCATPO transcript abundance was positively correlated with the ratio of betaxanthin to betacyanin in both green and red leaf sectors of A. tricolor. These data shows that the fourth group catalase, catalase-phenol oxidase, is present in plant, and might be involved in betaxanthin biosynthesis.Betalains are a group of nitrogen-containing pigments that color plants in most families of Caryophyllales. Their biosynthesis has long been proposed to begin with hydroxylation of L-tyrosine to L-DOPA through monophenolase activity of tyrosinase, but biochemical evidence  Amanita muscaria  and their subsequent oxidation to the corresponding o-quinones . Between two functions, the first one, i.e., monophenolase activity is known to be initial and rate-determining , but the monophenolase activity of purified tyrosinase toward L-tyrosine was much lower than its diphenolase activity to L-DOPA , a C4 betalain-producing plant. This new type of catalase, catalase-phenol oxidase (CATPO), might be involved in betalain biosynthesis, and in particular in betaxanthin biosynthesis.Keeping the second explanation in mind, we purified and biochemically characterized an enzyme with both catalase activity and tyrosinase activity from leaves of red amaranth (Amaranthus cruentus cv. Hopi Red Dye) were grown in greenhouse with natural light in summer, and common amaranth plants (Amaranthus tricolor) were purchased from a local market. Fresh young red-violet leaves of red amaranth were washed, cut and processed for enzyme and RNA extraction immediately, and fresh young tricolor leaves of common amaranth were washed, sectioned based on the color and separated for betalain content and quantitative Real-Time PCR (qRT-PCR) analysis.Red amaranth plants , a single lane of the gel was stained with 5 mM L-DOPA for 60 min at 45°C. Using the stained lane as a marker to the position of enzyme with diphenolase activity, the corresponding band was sliced out of the unstained lanes, excised and extracted in ice-cold 50 mM Tris-HCl buffer (pH 7.0). Purified enzyme (supernatant) was obtained by centrifugation and stored at −80°C. The enzymatic activities of the purified enzymes in different sets were detected according to in-gel staining of diphenolase activity  containing 10 mM EDTA, 10 mM mercaptoethanol, 0.1% (w/v) SDS, 0.1% (v/v) Triton X-100, and 40% (v/v) methanol. The samples were filtered through six layers of cheesecloth, centrifuged twice at 4°C at 8000 g for 30 min to remove plant debris. The supernatant (crude extract) obtained was stored at −80°C for further purification. The crude extract was purified through gel slice according to McMahon et al.  with smaSDS-PAGE was carried out on discontinuous gels  according to the method of Laemmli . Native −1 enzyme, and was placed at room temperature until reddish color appeared. A control (CK) was performed in parallel without the enzyme. Then the influence of cofactor L-DOPA on the reaction speed of monophenolase catalysis was tested according to Espín and Wichers (Agaricus bisporus. The reaction solution (200 μL) consisted of 50 mM potassium phosphate buffer (pH 7.0), 50 μM CuSO4, 0.2 mM L-tyrosine  in the Microplate Spectrophotometer PowerWave XS2 . Unless otherwise stated, experiments were repeated in triplicate. After determining the suitable cofactor concentration, the monophenolase activity was quantitatively determined spectrophotometrically by measuring dopaquinone accumulation at 475 nm  per minute, and the monophenolase activity toward L-tyrosine was calculated after removing the effect of L-DOPA as substrate.Monophenolase activity toward L-tyrosine of the purified enzyme was first qualitatively estimated by color change in reaction mixture. The reaction mixture (1 mL) contained 25 mM Tris-HCl buffer (pH 7.0), 2.5 mM L-tyrosine and 1 μg mL Wichers  with somKm value for L-tyrosine was estimated by measuring dopaquinone formation rate from L-tyrosine at concentrations of 0.1, 0.125, 0.15, 0.1675, 0.25, 0.3, and 1 mM under previous assay conditions.Effects of pH and temperature on the monophenolase activity of the purified enzyme were evaluated under the above assay condition. Optimal pH for the monophenolase activity was estimated by monitoring the enzyme's activity at pH range of 4.0–12.0. The buffer systems  used were acetate buffer for pH 4.0–5.0; phosphate buffer for pH 6.0–7.0; Tris-HCl buffer for pH 8.0–9.0; carbonate-bicarbonate buffer for pH 10.0–11.0; biphosphate-hydroxide buffer for pH 12.0. For determining the optimal temperature, the enzyme activity was measured at temperature ranging from 20 to 80°C. At the optimal temperature and pH, the 4. A control test was run in parallel in the absence of the inhibitor.Effects of six common tyrosinase inhibitors  on the monophenolase activity was evaluated under the previous assay condition without the addition of CuSO−1 enzyme. The reaction mixtures were incubated at the optimal temperature 60°C for 60 min and the absorbance was measured at 475 nm. The control was performed in parallel without the enzyme. One unit of the diphenolase of tyrosinase was defined as the amount of the enzyme that produces 1 μmol of dopaquinone per minute.Diphenolase activity of the purified enzyme was assayed with L-DOPA as substrate by measuring the dopaquinone accumulation at 475 nm. The reaction mixture (200 μL) contained 50 mM Tris-HCl (pH 8.0), 1 mM L-DOPA, and 2 μg mLKm value for L-DOPA was calculated by measuring dopaquinone formation rate from L-DOPA at eight concentrations  at the optimal temperature and pH.The effects of pH and temperature on the diphenolase activity were evaluated as on the monophenolase activity, except L-DOPA instead of L-tyrosine as the substrate. The The effects of five common tyrosinase inhibitors  on the diphenolase activity of the purified enzyme were evaluated through both in-gel staining and spectrophotometer. For in-gel staining determination, each three lanes of the gel, after electrophoresis on native PAGE with the purified enzyme (40 μL on every lane) were sliced out and incubated in 5 mM L-DOPA containing different inhibitors for 60 min at 45°C. A control test was run in parallel without the inhibitor. For spectrophotometry, the diphenolase activity was measured in the standard reaction medium in the presence or absence of the stated concentration of inhibitor.−1 using 0.1% formic acid  and 0.1% formic acid in acetonitrile . The HPLC linear gradient for separation was 1–90% (v/v). A spray voltage of 2 kV was applied. The MS scan range was mass-to-charge ratio 300–1800, and the 10 most intense precursor ions were selected for subsequent MS/MS scans. The MASCOT Server 2.4 (Matrix Science) software was used for MASCOT database searching. Peptide data were searched against the NCBI non-redundant database . The type of search was MS/MS Ion Search with 2+ as peptide charge and Viridiplantae (Green Plants) as taxonomy, and the instrument type was ESI-TRAP. Up to two missed cleavage was allowed, and mass tolerance for protein identification was 10 ppm for MS and 20 mmu for MS/MS. Peptides were considered identified if the MASCOT score was over the 95% confidence limit based on the “identity” score of each peptide.After SDS-PAGE, the band corresponding to the purified enzyme was excised from the gel, destained, and digested with sequencing-grade modified trypsin (Roche). Nano-LC-MS/MS analysis was conducted by using a Q-Exactive system (Thermo Scientific). The peptide mixture was separated using a nanoAcquity Ultra performance LC (Waters). A sample volume of 10 μL was loaded, and separation was performed at a flow rate of 200 nL minin vitro catalysis and inhibition assays. For catalysis assays, two methods were used: in-gel activity assay and spectrophotometer. After concentrating 100-folds by ultrafiltration  , and 2 μg mL−1 enzyme. Catalase unit, defined as the amount of enzyme that catalyzes the decomposition of 1 μmol H2O2 per minute, was calculated using a molar extinction coefficient of 39.4 M−1 cm−1 for H2O2 at 240 nm. For in vitro inhibition assays, the residual catalase activity was measured by spectrophotometer in the presence of each of four common catalase inhibitors  at 1 mM final concentration under the standard assay condition, and with absence of the inhibitor as control.Catalase activity of the purified enzyme was studied through e et al. . The rea2O2 as the substrate. At the optimal temperature and pH, the Km value for the catalase activity was determined using H2O2 at eight concentrations  under standard assay conditions.The optimal pH and temperature for the catalase activity was estimated as those for the tyrosinase activity except the temperature ranging from 10 to 60°C and HMesembryanthemum crystallinum catalase and Suaeda maritima subsp. salsa catalase to clone the cDNA of the purified enzyme. Total RNA was extracted from fresh young red-violet leaves of red amaranth with Trizol reagent (Invitrogen) following the manufacturer's protocols. First strand cDNA was obtained from 4 μg of the RNA using a PrimeScript II 1st strand cDNA Synthesis Kit (TaKaRa) according to the manufacturer's instructions. A core cDNA fragment was PCR-amplified with degenerate primers AcCATPOF1 and AcCATPOR1 , cloned into the pGEM-T vector (Promega) and sequenced at BGI Inc. (China) (The same for following PCR products). Subsequently, the 5′-end and 3′-end of the core sequence were PCR-extended with RACE technique by using Ex Taq™ DNA polymerase (TaKaRa), 5′/3′ Full RACE kit (TaKaRa), two 5′-end gene-specific primers  in GenBank by using itself as a query for phylogenetic analysis. The multiple sequence alignment was performed by using ClustalW2 and the unrooted tree was constructed with the software MEGA 6.0 using the neighbor-joining algorithm.A. tricolor were carefully separated and pooled respectively. The pooled sample of 200–300 mg (fresh weight) was homogenized to a fine powder in liquid nitrogen and then the betalains were extracted in 1 ml of 80% (v/v) methanol containing 50 mM sodium ascorbate as described in Harris et al.  of s et al. . BetacyaA. tricolor as described above. Approximately 1 μg RNA was digested using RNase-free DNase I (Promega) at 37°C and then reverse-transcribed to cDNA using a PrimeScript II 1st strand cDNA Synthesis Kit (TaKaRa). The qRT-PCR was conducted in a reaction mixture of cDNA, SYBR Green Master Mix (TaKaRa) and gene-specific primer pair  of ca. 10.5% and the specific activity of 0.0177, 0.2011, and 230.7 U mg−1 protein for monophenolase, diphenolase and catalase, respectively . Each of stained bands was gel-sliced out of the gel, and the second band was selected for further studies after preliminary test. This gel slice purification not only removed large amount of red–violet pigments betacyanins, but also gave an overall purification of 7.5-fold, with an overall activity yield of ca. 58 kDa on SDS-PAGE  and/or silver Figure , but the Figures  and abou Figures . These dAgaricus bisporus tyrosinase as positive control. Without cofactor, the purified enzyme displayed very low monophenolase activity toward L-tyrosine, whereas the mushroom tyrosinase showed much higher activity  on the monophenolase activity of the purified enzyme was investigated. As for classic tyrosinase, all inhibitors except tropolone, had a significantly inhibitory effect on the monophenolase activity Figure . Sodium e Chang, , displaye Chang,  showed ae Chang, .Km value of 0.2 mM toward L-tyrosine was calculated from the Lineweaver-Burk graphs for the purified enzyme  matched to with the highest score of 174 was 13%  analysis, due to lacking amino acid sequences of tyrosinases from betalain-containing plants and absence of genome information of 2O2 to produce oxygen and water as classic catalases (EC 1.11.1.6)  and L-DOPA in one reaction by the purified enzyme. Indeed, when the enzyme was added to the reaction mixture with both hydrogen peroxide (H2O2) and L-DOPA as substrates, the colorless reaction mixture became reddish in color and released bubbles (O2)  Aebi,  in one hAbove results showed that the purified enzyme possessed the catalase activity as classic catalases, in addition to its tyrosinase activity. Could this catalase activity be inhibited by common catalase inhibitors as classic catalases? To answer this question, we tested four common catalase inhibitors  at the optimal temperature and pH. Inhibition experiments showed that almost all inhibitors tested could inhibit the catalase activity of the purified enzyme to some extent Figure . Sodium These results of catalysis and inhibition experiments demonstrated that the purified enzyme indeed possessed the biochemical characteristics of classic catalases, and in particular of catalase-peroxidases, in addition to that of tyrosinases.Mesembryanthemum crystallinum and Suaeda maritima subsp. salsa whose amino acid sequences exhibited high identity with the peptide fragments of the purified enzyme, we first designed the degenerate primer pairs (Table KP710221) that consisted of 137 bp/335 bp of 5′/3′-UTR and a complete CDS which encoded a peptide of 492 amino acids deductively. Based on the cDNA, an entire CDS coding for 492 amino acids was cloned and identical to that of RACE cDNA. The predicted molecular weight of the deduced protein was 57 kDa, which was in agreement with that determined by SDS-PAGE.Both peptide fragment analysis and biochemical characterization revealed that the purified enzyme belonged to catalases but with tyrosinase activity. Accordingly, this implied that the purified enzyme would have both catalase active site and tyrosinase active site. To test this hypothesis, we cloned and analyzed the cDNA sequence of the purified enzyme. Based on the mRNA sequences of catalases from two betalain-producing plants, rs Table  to cloneBeta vulgaris subsp. maritima, M. crystallinum, and S. maritima subsp. salsa  in the deduced amino acid sequences of cloned cDNA. Therefore, we compared our amino acid sequences with those of some mammalian catalases that were reported to possess oxidase activity in the absence of hydrogen peroxide or any added cofactors  were typical only in catalases from betalain-producing plants or prevalent in non-betalain plant catalases. To answer this question, we performed bioinformatics analysis of relevant data in GenBank and AcCATPO possessed the monophenolase activity toward L-tyrosine and diphenolase activity to L-DOPA besides the catalase activity, so was it involved in betalain biosynthesis? With this question in mind, we examined the relation between the betalain content and the expression level of AcCATPO in green and red leaf sectors of A. tricolor. As shown in Figures AcCATPO transcript abundance revealed by qRT-PCR was about twice higher in green sectors than that in red ones, and it was positively correlated with the ratio of betaxanthins to betacyanins , the fourth group catalase which was verified by cloned cDNA. This ca. 170 kDa to apparent homogeneity by gel slice with L-DOPA as substrate from red-violet leaves of red amaranth, a betacyanin-producing C4 plant  and water  and subsequent oxidation of L-DOPA to produce dopaquinone  , we identified both catalase active site and possible phenol oxidase active site , but not monophenolic oxidation (monophenolase activity)   Figure . We obseIn summary, we identified, purified and characterized a catalase-phenol oxidase, AcCATPO, from red amaranth, a betalain-producing plant, and this Group 4 catalase had been only reported present in fungi and mammals including human  and Chinese High-Tech R&D Program “863” (Grant No. 2008AA10Z103) to X-GX.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer Mingyue Gou and handling Editor Chang-Jun Liu declared their shared affiliation, and the handling Editor states that, nevertheless, the process met the standards of a fair and objective review.


We experimentally studied the magneto-photocurrents generated by direct interband transition in InAs/GaSb type II superlattice. By varying the magnetic field direction, we observed that an in-plane magnetic field induces a photocurrent linearly proportional to the magnetic field; however, a magnetic field tilted to the sample plane induces a photocurrent presenting quadratic magnetic field dependence. The magneto-photocurrents in both conditions are insensitive to the polarization state of the incident light. Theoretical models involving excitation, relaxation and Hall effect are utilized to explain the experimental results. Recently, spin-polarized transport has been a main topic of spintronics. Optical injection has been widely used to generate a spin current ,2. In loMPE has been observed in InGaAs/InAlAs two-dimensional electron gas, GaAs/AlGaAs quantum well, graphene and so on -7. By coThe InAs/GaSb superlattice was fabricated by molecular beam epitaxy technique on semi-insulating (001)-oriented GaAs substrate. The 500-nm GaAs and 1,000-nm GaSb buffers were deposited on the substrate to relieve the lattice mismatch. Then an InAs/GaSb superlattice of 155 periods was deposited. The monolayer thicknesses of InAs and GaSb are 3.85 and 2.60 nm, respectively. The sample was not intentionally doped. The energy gap of this structure calculated by the k ·p theory is 129.5 meV. The standard Hall measurement demonstrates that the sample is n-type at room temperature, i.e. electrons are the main carriers contributing to transport. Since in the n-type superlattice spin relaxation time and lifetime of holes are much shorter than those of electrons, we neglect the contribution of holes to the magneto-photocurrents. Four pairs of ohmic contact electrodes which are parallel to  and [010] crystallographic directions are larger than that in the  and [010] crystallographic directions larger.By extracting the peak-to-peak values of the currents (nds (see ) seems urections . We specFor detailed analysis, the magnetic field direction dependence of the photocurrents can be well described by S1 and S1′) yield currents independent of the radiation polarization. The terms described by parameters S2, S2′ and S3, S3′ yield radiation linear polarization related currents proportional to |ex|2−|ey|2= cos(2α) and exey∗+eyex∗= sin(2α), respectively, where α is the angle between the plane of linear polarization and the x-axis. The terms proportional to the circularly polarized degree Pcirc (described by S4 and S4′) vanish for linearly polarized light excitation. I is the intensity of the incident light, it can be determined by light power per unit area of light spot. Bx=B0 cos(φ), By=B0 sin(φ), B0 = 0.1 T. φ is the angle between the magnetic field direction and [1C1 and C2 are background currents induced by the slight reduction of symmetry of the superlattice. The reduced symmetry is due to slight misorientation of substrate or presence of strain in the structure [x′∥ [100] and y′∥ [010]. Then the photocurrents can be described by  crystallographic direction. By rotating the magnetic field in the x-y plane, we obtained the dependence of the photocurrents on the magnetic field direction. As shown in Figure Jpp) in the two excitation conditions. In the [010] crystallographic direction, the ratio of Jpp under mid-infrared radiation excitation to Jpp under near-infrared radiation excitation is 0.58. In the [110] crystallographic direction, the ratio is 0.57. The magnetic field-induced current conversion efficiency for near-infrared excitation is nearly twice as that for mid-infrared excitation.In the above, we have discussed the magneto-photocurrents in the InAs/GaSb superlattice generated by direct interband transition. Here, we present the results of magneto-photocurrents generated by intersubband transition for comparison. We utilized a CO y-z and x-z planes. When the magnetic field is in the y-z plane, By=B0 cos(θ), Bz=B0 sin(θ) and Bx=0. θ is the angle between the magnetic field direction and the sample plane. The experimental results are presented in Figure In this section, we present results of a study of the magneto-photocurrents vs. the tilt angle of the magnetic field with respect to the sample surface. A linearly polarized 1,064-nm laser along -z was also used. The laser power was about 57 mW. The radiation linearly polarized direction was along the [100] and [010] crystallographic directions respectively when the magnetic field was rotated in the θ, sinθ and cosθ rather than by Equations 1 and 2. Thus, the mechanism of linear in-plane magnetic field-induced photocurrents (described by Equations 1 and 2) cannot hold here. Besides, the photocurrents cannot be explained by the mechanism of interplay of spin and orbit MPE observed in InSb/Sb quantum wells,  crystallographic direction, was normally irradiated on the sample plane. The laser power was about 62 mW. The variable magnetic field generated by an electromagnetic device was in the Jq on the strength of magnetic field are shown in Figure Jq presented clear quadratic magnetic field dependence. When the magnetic field was increased to 0.13 T, the current in [110] crystallographic direction increased by 17.35 pA; however, the current in [1The dependences of In summary, we have researched magneto-photocurrents in the InAs/GaSb superlattice when an in-plane and tilted magnetic field were applied respectively. The magneto-photocurrents in both conditions are insensitive to the polarization state of the incident light. A theoretical model involving anisotropic photo-excited carriers density is utilized to explain the in-plane magnetic field-induced MPE. Compared to the direct electron-photon interaction, the asymmetric electron-phonon interaction which contributes to the magneto-photocurrent may be more sensitive to the radiation polarization state. The quadratic magnetic field dependence of the magneto-photocurrents can be well illustrated by an additional Hall effect model.The authors declare that they have no competing interests.Y Li designed and carried out the experiments and wrote the manuscript. Y Liu and YC revised the paper. CJ, LZ, XQ and HG participated in the experiments. WM, XG and YZ designed and provided the sample. All authors read and approved the final manuscript.


Acanthamoeba strain UWC8 is an obligate amoeba endosymbiont belonging to the family of “Candidatus Midichloriaceae” in Rickettsiales. We report here the complete genome sequence of this bacterium, which should catalyze future studies of amoeba-symbiont interactions.The endosymbiont of  Candidatus Midichloriaceae” is a recently defined family in Rickettsiales that is distinct from previously described Rickettsiaceae, Anaplasmataceae, and Holosporaceae families . The cells were subsequently homogenized using a Dounce tissue grinder (Wheaton), and endosymbiont cells were isolated by differential centrifugation. The genome was sequenced by a 454 GS-FLX System using a 3-kb paired-end library, generating 104,222 reads totaling 31 Mbp with an average coverage depth of 20×. The genome was assembled by GS De Novo Assembler (Newbler) version 2.5.3. PCR and Sanger sequencing were used to close the gaps between contigs when necessary.The Acanthamoeba UWC8 consists of a single molecule of 1,615,277 bp with a G+C content of 34.7%. The genome was annotated using the IGS Annotation Engine (PfkA) converting beta-D-fructose-6P to beta-D-fructose-1,6-2P is missing. Instead, the fructose-1,6-bisphosphatase (fbp) catalyzing the opposite reaction is encoded in the genome, suggesting that gluconeogenesis instead of glycolysis is functional. Consistent with its obligate intracellular lifestyle, the endosymbiont is deficient in the biosynthesis of many of its own metabolic substances, including amino acids, nucleotides, and several coenzymes. Accordingly, it encodes a large number of ABC-type transporters and solute carrier families importing most of these nutrients from the host. The genome encodes complete pathways for synthesizing both peptidoglycan and lipopolysaccharide (LPS), suggesting that a complete cell envelope structure can be assembled. In addition, the genome encodes 4 genes in capsule biosynthesis and secretion that are absent in other Rickettsiales, which is consistent with the previous finding that capsules or slime layers are produced by this bacterium (The complete genome of the endosymbiont of n Engine : 89.5% oacterium . This geAcanthamoeba UWC8 has been deposited at DDBJ/EMBL/GenBank under the accession number CP004403.The complete genome sequence of the endosymbiont of


Arthritis Research & Therapy[+ T cells. From these results, the authors conclude that IFN type I cells co-act with Th17 cytokines in the pathogenesis of SLE. Surprisingly, they excluded CD25+ T cells from their analysis. In a previous study, we showed that Th cells from SLE patients expressing CD25med and CD25high are also able to produce IFN-γ and IL-17A [+CD25+ T cells from IFN type I-positive and IFN type I-negative SLE patients. Furthermore, it should be proven that the genetic signature is solely responsible for the increased IFN production by Th cells. In addition, their finding that CCR6+ T cells are capable of producing IL-21 indirectly confirms our previous observation that IL-17+ T cells are a main source of IL-21 in patients with SLE [We read with great interest the article by Brkic and colleagues in a recent issue of & Therapy. In thatwith SLE . PossiblFinally, we agree that there might be a co-activity between IFN-I- and IL-17-producing cells as described by Brkic and colleagues. However, considering our findings that T cells with a regulatory phenotype are able to produce IFN-γ and IL-17A in patients with SLE, we suggest that primarily the plasticity of T cells is altered in patients with SLE .IFN: Interferon; IL: Interleukin; SLE: Systemic lupus erythematosus; Th: T helper.The authors declare that they have no competing interests.All authors contributed to the interpretation of data. SD drafted the manuscript. All authors read and approved the final manuscript.


Methoxy-modified kaolinite was used as a novel carrier for loading and release of the herbicide 3-amino-1,2,4-triazole, known as amitrole (abbreviated here as AMT). The methoxy modification made the interlayer space of the kaolinite available for AMT intercalation. The AMT loading content in methoxy-modified kaolinite reached up to 20.8 mass% (twice the loading content by unmodified kaolinite). About 48% of this amount is located in the interlayer space. The release profiles of the AMT fit with the modified Korsmeyer-Peppas model. Due to the diffusional restriction of the intercalated AMT by the lamellar structure of the kaolinite and the strong electrostatic attraction between the intercalated AMT and the kaolinite, a slow release of AMT from the methoxy-modified kaolinite was achieved. These results show that the methoxy-modification is a facile method to make the interlayer space of kaolinite available for hosting other guest molecules. The methoxy-modified kaolinite is a promising candidate for high-capacity loading and controlled-release of other molecules such as drugs, agrochemicals, and biochemicals. Pesticides, mainly insecticides, herbicides, and fungicides, are used worldwide to increase global agricultural productivity, to reduce insect-borne endemic diseases, and to protect plantations2via inhibition of carotenoid biosynthesisAmitrole  is a non-selective polar herbicide with a wide spectrum of activity against annual and perennial broadleaf and grass-type weeds, acting 2(OH)4 octahedral sheet and SiO4 tetrahedral sheet. The adjacent sheets are connected together by apical oxygen to form a layer with ideal chemical formulae Al2Si2O5(OH)4. The kaolinite layers are linked by hydrogen bonding. Conventionally, kaolinite is rarely used as a carrier for organic compounds such as herbicides despite its much lower cost than most commercialized carriers such as synthetic polymers, because of its low cation exchange capacity (CEC less than 10 mmol/100 g)2/g)10Kaolinite is a 1:1 clay mineral consisting of AlOAt our knowledge, the methoxy-modified kaolinite was used for the first time as carrier for the loading and release of the intercalated AMT herbicide. Attention was focused on the mechanism of the methoxy-modification on the AMT loading and on its release from kaolinite.d001 value of 0.71 nm  of 0.85 nm , the d001 value of kaolinite remains unchanged (MeOH)  in the interlayer space. However, due to the intercalation of AMT in the interlayer space of the methoxy-modified kaolinite, the AMT loading content in AMT-KaolMeOH, calculated from the percentage of N, reached up to 20.8 mass%, which is twice the loading content of AMT-Kaol (10.3 mass%).The raw kaolinite (Kaol) shows a typical diffraction pattern with a characteristic  0.71 nm . Graftin 0.85 nm . The remnchanged , indicatd (MeOH) , the d00d value of 0.53 nm is assigned to the (020) crystallographic plane of AMT crystallite (JCPDS card No. 44-1816). This reflection is used to calculate the mean diameter (D) of AMT crystallites from the Scherrer equation. The calculated D values of the AMT crystallites in AMT-Kaol and AMT-KaolMeOH are 31.4 and 33.4 nm, respectively. Sliwinska-Bartkowiak et al.In the XRD patterns of the AMT-loaded kaolinite , the ref−1, as well as the stretching vibration of the inner hydroxyl groups at 3621 cm−1. In the DRIFT spectrum of KaolMeOH   . This obMeOH  from 180 to 340°C is attributed to the dehydration of the interlayer water and the decomposition of the non-intercalated AMT adsorbed on the external surface of kaolinite. The second mass loss (LossII) from 340 to 500°C (endothermic peak at 371.4°C in the DSC curve) probably resulted from three simultaneous thermal phenomena: i) the loss of the grafted methoxy groups, ii) the dehydroxylation of kaolinite, and iii) the decomposition of the intercalated AMT. The decomposition temperature of the intercalated AMT (340 ~ 500°C) is higher than the decomposition temperature of the non-intercalated AMT (180 ~ 340°C), indicating that AMT is more thermally stable when it is intercalated.In the TG curve of AMT-KaolMeOH , two steMeOH, the respective mass losses of the non-intercalated AMT (from 180 to 340°C) and of the intercalated AMT (from 340 to 500°C) did not overlap. Also, the decomposition of the residual non-intercalated AMT between 500 and 750°C  of 0.50, while an anomalous transport (non-Fickian) mechanism is indicated by an n value of 0.50 ~ 1.0. The parameters of this equation for each AMT release profile and the corresponding coefficients of determinations (2R) are listed in 2R (> 0.989) suggest that the release profiles of AMT were well fitted using the modified Korsmeyer-Peppas model. The a value for AMT-KaolMeOH is 0.11, much smaller than the a value for AMT-Kaol (0.68), indicating a much slower release of AMT from AMT-KaolMeOH than that from AMT-Kaol. The n values for AMT-Kaol is 0.36, lower than 0.50, indicating that some other release mechanism occuredn value for AMT-KaolMeOH is 0.79, indicating a non-Fickian diffusion mechanism. All these results suggest that the methoxy modification of the kaolinite influence the release mechanism of AMT.The release kinetics of AMT from kaolinite samples was evaluated by fitting the release profiles to the modified Korsmeyer-Peppas model18The intercalation of AMT in the methoxy-modified kaolinite leads to an interlayer distance of 0.30 nm which was slightly smaller than the height of AMT with molecular dimensions approximately 0.75 nm × 0.62 nm× 0.37 nm (length × width × height)The intercalated amorphous AMT exhibited higher thermal stability than the non-intercalated crystalline AMT because the intercalated AMT i) formed strong electrostatic attraction with kaolinite, and ii) could be protected from the thermal decomposition by the lamellar structure of the kaolinite. This finding is in agreement with the higher thermal stability of other intercalated organic guests that have been found to decompose at higher temperatures in the interlayer space of clay minerals, such as cationic surfactant in montmorilloniteKa value of 4.1 and is not positively charged in a wide range of pH. So, on montmorillonite it is difficult to load AMT through cation exchange but AMT was primarily adsorbed as neutral molecules and exhibited a low level of loadingThe methoxy-modification allowed the intercalation of AMT in the interlayer space of kaolinite, and significantly promoted the loading capacity till 20.8 mass%. This value is much higher than the AMT loading that has been achieved on montmorillonite (1.5 mass%)MeOH also exhibited a controlled-release of AMT, which was achieved via the two previously indicated routes: the diffusional restriction of the intercalated AMT by the lamellar structure of kaolinite and the strong electrostatic attraction of the intercalated AMT to kaolinite. Generally, tubular halloysite, chemically and structurally similar to kaolinite, is used as carrier for loading and release of various guests2728293031323327Moreover of the high-capacity loading of AMT, AMT-KaolPrevious results and discussion show that the promotion of the loading and controlled-release of AMT by methoxy-modification on kaolinite succeeded to act with the following mechanism. The methoxy-modification introduced a plane of methoxy groups in the interlayer space of kaolinite weakening the original hydrogen bonding between the adjacent layers. This interlayer space became available for further AMT intercalation. Moreover, the lamellar kaolinite structure could significantly restrict the diffusion of the intercalated AMT, leading to its slow release from the modified kaolinite.via the slow release of these anticorrosives or antimicrobials agents. Other chemicals such as drugs, agrochemicals, and biochemicals of similar functional groups and molecular dimensions to AMT could also achieve high loading and controlled release in methoxy-modified kaolinite.The methoxy-modified kaolinite is a potentially promising carrier for the long-term controlled release of certain specific guest molecules, enabling promising prospects in future applications. For example, because kaolinite is widely used in paint, the intercalation of some anticorrosives (benzotriazole or 8-hydroxyquinoline) or antimicrobials into the interlayer space of the kaolinite could possibly endow the paint with long-acting anticorrosion or antimicrobic functions In summary, the methoxy modification of kaolinite could develop an additional interlayer space for loading of pesticides, drugs, and some other bioactive chemicals. The methoxy-modified kaolinite loaded with these chemicals also allowed their controlled release.Amitrole (AMT) was purchased from Meryer (99%). All other reagents were of analytical grade. A high-purity kaolinite sample, obtained from Guangdong Province, China, was used as received without further purification and labeled as Kaol.via centrifugation and then stored in a wet state for further use. The methoxy-modified kaolinite was labeled as KaolMeOH.Dimethyl sulfoxide (DMSO) was first intercalated into the interlayer space of Kaol as previously reportedvia centrifugation and dried overnight at 80°C. The AMT-loaded kaolinite samples were identified by adding the prefix “AMT-” to the starting materials; for example, AMT-KaolMeOH refers to the kaolinite that was modified with MeOH and then loaded with AMT.A sample of 2.0 g of AMT was dissolved in 20 mL of MeOH, and approximately 1.0 g of each used clay mineral, before and after methoxy modification, was added under constant stirring for 24 h at room temperature. The solid part of the dispersion was separated The AMT-release tests were conducted using an RCZ-8M dissolution tester  following a paddle method. Approximately 0.5 g of AMT-loaded clay mineral sample was sealed in a dialysis bag (molecular mass cutoff: 3500 Da), and then soaked in 500 mL of distilled water at room temperature with a rotation speed of 100 rpm. At suitable intervals, 5 mL of the dissolution medium was withdrawn, and an equivalent volume of fresh medium was added. The AMT content was determined at 239 nm using a PerkinElmer LAMBDA 850 UV/Vis spectrophotometer. Each dissolution test was performed in triplicate. After the achievement of 100% release of AMT, the clay mineral samples were collected for thermal analysis to calculate the amount of the intercalated AMT.The X-ray diffraction (XRD) patterns were obtained using a Bruker D8 Advance diffractometer with a Ni filter and Cu Kα radiation (λ = 0.154 nm) generated at 40 kV and 40 mA. Diffuse reflectance infrared Fourier-transform (DRIFT) spectra were obtained using a Bruker Vertex 70 Fourier-transform infrared spectrometer at room temperature. The CHN elemental analyses were performed using an Elementar Vario EL III Universal CHNOS Elemental Analyzer. Thermogravimetry (TG) and differential scanning calorimetry (DSC) were performed with a Netzsch STA 409PC instrument. Transmission electron microscopy (TEM) observations were recorded using a 200 kV JEOL JEM-2100 high-resolution transmission electron microscope. Nitrogen adsorption-desorption isotherms were measured using a Micromeritics ASAP 2020 instrument at liquid nitrogen temperature.D.Y.T. and P.Y. conceived and designed the experiments. D.Y.T. and D.L. analyzed the data. D.Y.T. and P.Y. wrote the manuscript. F.A.B. and H.P.H. are involved in the related discussion and F.A.B. helps to improve the quality of the manuscript.SI


Supplemental Digital Content is available in the text Adjuvant chemotherapy is recommended for postoperative stage II-IIIB nonsmall cell lung cancer patients. However, its effect remains controversial in stage IB patients. We, therefore, performed a meta-analysis to compare the efficacy of adjuvant chemotherapy versus surgery alone in stage IB patients.Six electronic databases were searched for relevant articles. The primary and secondary outcomes were overall survival (OS) and disease-free survival (DFS). The time-to-event outcomes were compared by hazard ratio using log-rank test.Sixteen eligible trials were identified. A total of 4656 patients were included and divided into 2 groups: 2338 in the chemotherapy group and 2318 in the control group (surgery only). Patients received platinum-based therapy, uracil-tegafur, or a combination of them. Our results demonstrated that patients can benefit from the adjuvant chemotherapy in terms of OS (HR 0.74 95% CI 0.63–0.88) and DFS (HR 0.64 95% CI 0.46–0.89). Patients who received 6-cycle platinum-based therapy (HR 0.45 95% CI 0.29–0.69), uracil-tegafur (HR 0.71 95% CI 0.56–0.90), or a combination of them (HR 0.51 95% CI 0.36–0.74) had better OS, but patients who received 4 or fewer cycles platinum-based therapy (HR 0.97 95% CI 0.85–1.11) did not. Moreover, 6-cycle platinum-based therapy (HR 0.29 95% CI 0.13–0.63) alone or in combination with uracil-tegafur (HR 0.44 95% CI 0.30–0.66) had advantages in DFS. However, 4 or fewer cycles of platinum-based therapy (HR 0.89 95% CI 0.76–1.04) or uracil-tegafur alone (HR 1.19 95% CI 0.79–1.80) were not beneficial.Six-cycle platinum-based chemotherapy can improve OS and DFS in stage IB NSCLC patients. Uracil-tegafur alone or in combination with platinum-based therapy is beneficial to the patients in terms of OS, but uracil-tegafur seems to have no advantage in prolonging DFS, unless it is administered with platinum-based therapy. Though surgery is regarded as the primary treatment modality for early stage NSCLC, only 20% to 25% of the tumors are suitable for potentially curative resection, and a substantial percentage of these patients eventually develop local recurrence or distant metastases. As a result, more effective treatment strategies to reduce lung cancer mortality and recurrence rates are needed.Roughly 1.5 million new cases of lung cancer are diagnosed worldwide each year5 and meta-analyses.7 Most of the randomized clinical trials reported positive results in patients with completely resected stage IB, II, and IIIA NSCLC.5 Only 1 large randomized trial CALGB96338 focused on completely resected stage IB (T2N0) patients. However, its final results of overall survival (OS) and disease-free survival (DFS) lacked statistical significance.Five-year survival improvements of 5% to 10% have been reported with cisplatin-based adjuvant chemotherapy from multiple large randomized clinical trialsCurrently, the role of adjuvant cisplatin-based chemotherapy has been established by multiple large randomized phase III trials for resected stage II and IIIA NSCLC, but its role is controversial in stage IB patients. We, therefore, carried out a systematic review and meta-analysis to provide more reliable and up-to-date evidence on the effect of postoperative chemotherapy in stage IB patients through OS and DFS to identify whether the effect varies by patient subgroup. This included trying to verify the effects of different regimens and duration of postoperative chemotherapy.The electronic search was performed using PubMed, Medline, Cochrane Central Register of Controlled Trial, Cochrane Database of Systematic Reviews, ACP Journal Club, and Database of Abstracts of Reviews of Effects from the date of the earliest publication (1962) to October 2014. In order to achieve the maximum sensitivity, we used the following search strategy: “lung cancer” [all fields] AND . Discrepancies between reviewers were resolved by the discussion and consensus with the senior investigators . The publication characteristics and time-to-event data including median overall survival hazard ratio (HR), disease-free survival HR, and 95% confidence interval (CI) were reviewed and extracted. Some of the included publications did not report the HR directly but they reported Kaplan–Meier curve or its 2 tests were conducted to access the fit of distribution.10 In order to minimize this interference induced by the number of studies, I2 test was used to estimate the variation across the studies instead of the Q test,10 because Q test results were closely related to the number of included studies. The I2 was calculated by the formula: I2 = 100% × (Q−df)Q; Q stood for a heterogeneity statistic and df was defined as the degree of freedom . The heterogeneity was defined as low (25%–49%), moderate (50%–74%), or severe (>75%). Fixed-effect analysis model was used to calculate the HR. If the heterogeneity was severe, a random-effects analysis model would be used. In addition, the sensitivity test or subgroup analysis would be performed. Z-test was performed to calculate the P value, which was 2-sided and defined as statistically different when P < 0.05. The statistical analysis was conducted via Review Management 5.2 and Stata 12. The publication bias was analyzed via Stata 12. It would be considered insignificant when the P > 0.05 in both Egger and Begg tests.12A systemic review and meta-analysis were performed to compare the postoperative chemotherapy to surgery alone by estimating the HR of OS and DFS. The data for analysis were categorical. Based on the null hypothesis stating that the frequency distribution of certain events observed in a sample were consistent with a particular theoretical distribution, χ27 were chosen for data extraction and meta-analysis ” and “N/M” . However, 2 articlesP value was 0.038. We performed a sensitivity analysis and found that no publication bias existed either in Begg test or Egger test after excluding the data of Park's27 study. We decided to exclude Park's data from the analysis. There were 16 articles in the final analysis  group.16 In 5 studies from Japan, 884 patients were assigned to chemotherapy treatment group and received uracil-tegafur alone.19 Furthermore, 242 patients in 3 studies had received a combination of platinum-based therapy and uracil-tegafur.26A total of 4656 patients were eligible for the meta-analysis. Among them, 2338 patients were assigned to the adjuvant chemotherapy group and 2318 were assigned to the control group . Characteristics of patients are demonstrated in Tables P < 0.001). In the platinum-based chemotherapy group, an interesting result was observed. Patients who received 6 cycles of platinum-based therapy had better OS than those in the control group (HR 0.45 [95% CI 0.29–0.69] P < 0.001). However, patients who received 4 or fewer cycles of platinum-based therapy had no significant superiority in OS compared to the control group (HR 0.97 [95% CI 0.85–1.11] P = 0.664). In the uracil-tegafur oral chemotherapy subgroup, the treatment group had an obvious better overall survival rate than the control group (HR 0.71 [95% CI 0.56–0.90] P = 0.004). Patients who received the combination of platinum-based chemotherapy and uracil-tegafur also had a better OS than those in the control group (HR 0.51 [95% CI 0.36–0.74] P < 0.001) . There was a slight heterogeneity overall , even though the random-effect model was selected. By performing the sensitivity analysis, we discovered that the results were consistent with the previous results after discarding the data that contributed the most heterogeneity.As shown in Figure P = 0.149). However, the DFS of the 6-cycle platinum-based therapy subgroup was significantly better than the control group (HR 0.29 [95% CI 0.13–0.63] P = 0.002). The platinum-based therapy in combination with uracil-tegafur treatment was superior to the control group (HR 0.44 [95% CI 0.30–0.66] P < 0.001). But patients who received only uracil-tegafur had no advantage compared to the patients in the control group (HR 1.19 [95% CI 0.79–1.80] P = 0.399) , except for the 6-cycle platinum-based group . The overall heterogeneity was rather significant . Nevertheless, our results showed that adjuvant chemotherapy had a slight advantage (HR 0.64 [95% CI 0.46–0.89] P = 0.008).Even though heterogeneity was not existent in any of the subgroups , it may not be a definite conclusion. Even so, we discovered some valuable information through the subgroup analysis process.A total of 16 randomized trials were analyzed to determine via OS and DFS whether or not adjuvant chemotherapy is beneficial for stage IB completely resected NSCLC patients. Our analysis found adjuvant chemotherapy to be beneficial in patients with completely resected stage IB NSCLC in terms of OS and DFS. However, due to the overall heterogeneity of OS and DFS . It indicated that 4 or fewer cycles of adjuvant chemotherapy could bring benefit to the stage II-III NSCLC patients.Moreover, we did an analysis of the same endpoints on postoperative stage I-III NSCLC patients. The results demonstrated that patients in both 6-cycle and fewer cycle subgroups gained benefits from the adjuvant chemotherapy. Data were shown in supplement , the head-to-head comparison between 6-cycle and fewer cycle adjuvant chemotherapy was lacking. More clinical trials have to be performed to compare the effect of different cycles of platinum-based adjuvant chemotherapy in long-term survival.The HRs reported in the 6-cycle subgroup were for 10-year OS and DFS; HRs in other included articles presented only 5-year OS and DFS. It is possible that both subgroups have no advantage when comparing the 5-year OS or DFS to surgery alone, but have more long-term benefits such as prolonging 10-year OS and DFS. Although Mineo et al37 After that, more studies were conducted and it was discovered that oral administration of uracil-tegafur had an anticancer effect in several solid cancers. The West Japan Study Group for Lung Cancer Surgery reported that OS was longer in the treatment group (uracil-tegafur) patients than in patients assigned to the control  group after complete resection of stage I-III lung cancer.24 Our study results were the same as these previous studies for patients with resected stage IB NSCLC who received uracil-tegafur, except that there was no benefit in terms of DFS.Oral treatment of uracil-tegafur was firstly introduced to the public in 1979 by Japanese researchers.16 They reported that patients in both treatment arms had better outcomes than those in the control arm. In terms of the comparison between 2 treatment arms, it demonstrated that there was a slight advantage of uracil-tegafur plus platinum-based therapy, but not statistically significant. However, we were unable to confirm this finding through our own subgroup analysis because our own analysis only compared platinum-based chemotherapy combined with uracil-tegafur to our control group as there was not enough data present. Future studies are needed to explore the role of uracil-tegafur in combination with platinum-based therapy for patients with NSCLC.In 3 trials of uracil-tegafur plus platinum-based therapy in patients with stage IB lung cancer, the patients assigned to the treatment group had better OS and DFS than those assigned to the control group. The study reported by Imaizumi et al is a 3-arm trial containing uracil-tegafur, uracil-tegafur plus platinum-based therapy, and control groups.P value of log-rank test. This method is not as accurate as the HRs reported by the authors. Secondly, the OS and DFS were calculated from the time after surgery in some studies, but from the date of diagnosis in other studies, which might prolong the OS. Thirdly, during the searching and evaluating process, we noticed that some studies included patients other than stage I; the data of stage I patients were unable to be separated from the data as a whole and were therefore discarded in accordance with our exclusion criteria. The loss of such data potentially weakens the reliability of our analysis results because of the loss of such data. Next, the OS and DFS Kaplan–Meier curves of all included patients were unable to be presented without the individual data. We analyzed the OS and DFS between different chemotherapy subgroups. If the individual data were available, more subgroup analysis could be performed, for example, gender, tumor size, age, toxicity, and smoking condition, which would add to precision of our study. Finally, all the studies of uracil-tegafur were performed on Asian people; other races were not included. Thus, a conclusive claim that uracil-tegafur is beneficial to all postoperative stage IB NSCLC patients is not able to be made.A number of limitations have to be addressed in the present study. Firstly, some of the HRs were not directly reported in the texts so we had to calculate the HR from the observed patient numbers and In conclusion, platinum-based therapy is able to increase long-term OS and DFS of postoperative stage IB NSCLC patients. Patients who received postoperative 6-cycle of platinum-based chemotherapy, uracil-tegafur, or a combination of the 2 had better OS than the patients in the control group. However, patients who received only 4 or fewer cycles of platinum-based chemotherapy had no advantage comparing to the control group. For DFS, postoperative 6-cycle platinum-based chemotherapy alone or in combination with uracil-tegafur brought benefit to patients compared to the surgery group, but patients who were prescribed 4 or less cycles of platinum-based chemotherapy or uracil-tegafur alone had no advantage in prolonging DFS compared to the control group. Though a number of limitations exist in the present study, our findings provide potentially useful information; however, new randomized control trials are needed to make a more conclusive argument and to figure out the different effect of 6 cycles and fewer cycles of platinum-based chemotherapy and explore the effect, dosage, and toxicity of the regimens.


The potential effects of amalgam fillings on the development of Alzheimer’s disease (AD) are not well understood. The aim of the study was to evaluate the association between dental amalgam fillings and Alzheimer’s disease in Taiwanese population aged 65 and older.Data were retrieved from the Longitudinal Health Insurance Database (LHID 2005 and 2010). The study enrolled 1,943,702 beneficiaries from the LHID database. After excluding death cases and individuals aged 65 and under, 207,587 enrollees were finally involved in the study. Dental amalgam fillings are coded as 89001C, 89002C, 89003C, 89101C, 89102C, or 89103C in the national health insurance research database (NHIRD). Alzheimer’s disease was diagnosed using the International Classification of Diseases, Ninth Revision, Clinical Modification (ICD-9-CM) codes 331.0.Individuals exposed to amalgam fillings had higher risk of Alzheimer’s disease  than their non-exposed counterparts. Further analysis showed that the odds ratio of Ahlzheimer's disease was 1.07 (95 % CI = 0.962-1.196) in men and 1.132 (95 % CI = 1.022-1.254) in women.Women who were exposed to amalgam fillings were 1.132 times more likely to have Alzheimer’s disease than were their non-exposed counterparts. Amalga is indicated when early caries are present. Crowns are indicated when the tooth structure is severely destroyed possibly due to severe caries while bridges are indicated in edentulous areas. (2) Most of the crowns and bridges are porcelain-fused-metal. That is, they are covered by porcelain with a natural-tooth-like look. The degree of metal exposure to the oral cavity is less. Even with all-metal crowns and bridges, the surfaces of the prosthesis are highly polished to decrease the abrasion of the metals. Therefore, effects due to oral exposure to nickel, palladium, chromium or cobalt were possibly minimized.After adjusting for age, income and residential region, women exposed to mercury amalgam fillings were 1.132 times more likely to have AD than were their non-exposed counterparts.